Pluripotent cells

ABSTRACT

Pluripotent cells that are immunopositive for both the neural progenitor marker nestin and a pluripotent cell marker are provided. The cells exhibit rapid doubling times and can be maintained in vitro for extended periods. Also provided are cell cultures containing the pluripotent cells, a method of transplanting human pluripotent cells to a host, and a method of reducing seizure activity in a subject. These pluripotent cells, when transplanted into the ventricle of a host animal, migrate to the site of damage and adopt a suitably corrective phenotype, resulting in both structural and functional restoration.

This application is a divisional of U.S. patent application Ser. No.11/755,224, filed May 30, 2007, which claims the benefit of provisionalpatent application No. 60/803,619, filed May 31, 2006; and is acontinuation-in-part of U.S. patent application Ser. No. 11/002,933,filed Dec. 2, 2004, which claims priority to provisional application No.60/526,242, filed Dec. 2, 2003, the entire contents of each of which areincorporated by reference herein. Throughout this application variouspublications are referenced. The disclosures of these publications intheir entireties are hereby incorporated by reference into thisapplication in order to describe more fully the state of the art towhich this invention pertains.

BACKGROUND OF THE INVENTION

Parkinson's disease (PD) is a very common neurodegenerative disorderthat affects more than 2% of the population over 65 years of age. PD iscaused by a progressive degeneration and loss of dopamine (DA)-producingneurons, which leads to tremor, rigidity, and hypokinesia (abnormallydecreased mobility). It is thought that PD may be the first disease tobe amenable to treatment using stem cell transplantation. Factors thatsupport this notion include the knowledge of the specific cell type (DAneurons) needed to relieve the symptoms of the disease. In addition,several laboratories have been successful in developing methods toinduce embryonic stem cells to differentiate into cells with many of thefunctions of DA neurons.

While attempts have been made to propagate neural progenitor cells(partially differentiated precursors to neurons and glial cells) for usein neurotransplantation and for drug screening, these efforts have metwith limited success. Neurobasal medium has allowed for fast doublingtimes of cultured neural progenitor cells, but these doubling times areobserved for about one month, after which the cells differentiate andlose their progenitor phenotype. Typically, with the most optimalculture conditions, neural progenitor cells will survive for only about10 passages in culture. In addition, only about 1-2% of neuralprogenitor cells survive cryopreservation. Moreover, current efforts tomaintain neural progenitor cells in vitro require the use of a feederlayer and/or introduce animal components. Even with use of a feederlayer, neural progenitor cells have been maintained for only about 6months. For clinical applications, it is desirable to obtain andmaintain human neural progenitor cells that are free of animalcomponents and do not require the use of a feeder layer.

Likewise, the development of stem cells for transplantation has beenapproached with an assumption that such pluripotent cells would have tobe genetically modified in order to express the desired phenotype fortherapeutic benefit, such as the neurotransmitter dopamine for treatmentof Parkinson's disease. Alternatively, it has been assumed that one mustexpose the stem cells to conditions that would induce predifferentiationto achieve the desired phenotype.

There remains a need for a large quantities of undifferentiated neuralprogenitor cells and pluripotent or totipotent stem cells fortransplantation and for drug screening, particularly for humanprogenitor and stem cells. A need also exists for cells that are capableof long-term proliferation in vitro. In particular, there is a need formethods of maintaining and propagating neural progenitor and pluripotentcells for extended periods of time, and for methods that optimize yieldfollowing cryopreservation.

SUMMARY OF THE INVENTION

The invention provides pluripotent cells that can be propagated andmaintained for extended periods of time in culture in the absence of afeeder layer. Surprisingly, these cells express both markers forpartially differentiated neural progenitor cells (nestin, SSEA-1) andmarkers for pluripotent stem cells (TRA-1-60, TRA-1-81, SSEA-4, Oct-4).Also provided are methods of propagating and using such cells. Thesecells are useful for transplantation to hosts having disease and/ordamage, particularly of the central nervous system, as they are capableof migrating to the sites in need of repair, and of adopting a phenotypemost appropriate to the nature of the damage or disease. Moreover, thepluripotent cells of the invention have been found to have a surprisingability to protect against massive structural and functional damage.

The pluripotent nature of these cells renders it unnecessary togenetically modify the cells to be transplanted, and also obviatesconcerns about selecting the appropriate phenotype of cells, orpredifferentiating cells prior to transplantation. Accordingly, theinvention provides, in one embodiment, a substantially pure culture ofpluripotent cells that is free of genetically modified cells. Use ofthese pluripotent cells provides particular advantages fortransplantation and therapy over, for example, use of predifferentiatedcells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the growth of cultured NPC in low calcium(0.06 mM) EMEM supplemented with (“E+”) various combinations of EGF (E),bFGF (F), TGFα (T) and LIF (L). E+EFT provided optimal growth of NPC insuspension.

FIG. 2 is a graph showing the growth of cultured NPC in Neurobasal™medium supplemented with (N+) various combinations of EGF (E), bFGF (F),TGFα (T) and LIF (L). N+EFT provided optimal growth of attached cells.Growth rates declined, however, after 3-4 months in vitro.

FIG. 3 is a photomicrograph showing immunohistochemistry of T and Mbrain progenitor lines. A strong BrDU-positive reaction was observed inthe M5 line cells after 138 passages. 20× magnification.

FIG. 4 is a phase contrast photomicrograph that shows a confluent growthof M5 NPC cells. Almost all cells maintain undifferentiated condition.10× magnification.

FIG. 5 is a phase contrast photomicrograph that shows a typical“embryoid body” formed by the brain progenitor cells and characteristicfor stem/progenitor cells. 10× magnification.

FIG. 6 is a phase contrast photomicrograph that shows brain progenitorcells from the 5^(th) passage of T5 line growing in small floatingclusters. 10× magnification.

FIG. 7 is a phase contrast photomicrograph that shows a small floatingcluster of the NPC and a number of the NPC cells that are gettingattached to the culture flask due to the increase in medium Ca++concentration from 0.05 mMol to 0.1 mMol. 10× magnification.

FIG. 8 is a phase contrast photomicrograph that shows the NPC from T5line growing as embryoid bodies. 154^(th) passage. 10× magnification.

FIG. 9 is a photomicrograph showing a flat cluster of the NPC from M5line. Ca++ concentration of the culture medium at 0.1 mMol. 46% of thecells are BrDU-positive. 20× magnification.

FIG. 10 is a photomicrograph showing a large floating cluster of cellsfrom T5 line, with a mitotic figure in the center. Giemza stain. 40×magnification.

FIG. 11 is a photomicrograph showing the tyrosine hydroxylase(TH)-positive NPCs in the striatum of a 6-OHDA lesioned rat. 20×magnification.

FIG. 12 is an electron micrograph showing the ultrastructure of anundifferentiated NPC from T5 line. 13,000× magnification.

FIG. 13 is an electron micrograph showing the ultrastructure of a NPCfrom M5 line. Its cytoplasm contains many mitochondria. 13,000×magnification.

FIG. 14 is a photomicrograph showing bromodeoxyuridine (BrDU)immunopositive NPC in a M5 line suspension. Immunoreactive cells stainedwith diaminobenzidine (DAB). 40× magnification.

FIG. 15 is a photomicrograph showing bromodeoxyuridine (BrDU)immunopositive NPC in a M3 single cell suspension. Immunoreactive cellslabeled with fluorescein. 20× magnification.

FIG. 16 is a photomicrograph showing nestin immunopositive NPC in a M3single cell suspension. Immunoreactive cells labeled with fluorescein.20× magnification.

FIG. 17 is a photomicrograph showing co-expression of nestin and Oct-4in the same NPCs, green fluorescence representing Oct-4 and redrepresenting nestin. 20×.

FIG. 18 is a photomicrograph showing an amber-brown human neuron withthe branching extensions at the center of the picture and a glial cellat the right lower corner of the picture in the rat putamen. These cellsmigrated from the cerebral ventricle of the animal that showed a 70%improvement in its rotational behavior 4 months after theintraventricular injection of 500,000 undifferentiated brain progenitorcells. Anti-human mitochondrial antibodies. 40×

FIG. 19 is a series of photomicrographs showing doublestaining of PC fornestin (center panel; Texas red) and TRA-1-60 (right panel; FITC).Nuclei are stained with DAPI (left panel) to reference cell location,size and shape. The lower panel shows all 3 images overlaid, indicatingthat most cells express characteristics of both embryonic,undifferentiated cells (TRA-1-60) and partially differentiated neuralprogenitor cells (nestin) simultaneously. 40×

FIG. 20 is a series of photomicrographs showing doublestaining of PC fornestin (center panel; Texas red) and TRA-1-81 (right panel; FITC).Nuclei are stained with DAPI (left panel) to reference cell location,size and shape. The lower panel shows all 3 images overlaid, indicatingthat most cells express characteristics of both embryonic,undifferentiated cells (TRA-1-81) and partially differentiated neuralprogenitor cells (nestin) simultaneously. 40×

FIG. 21 is a series of photomicrographs as in FIGS. 19 and 20, exceptwith SSEA-4 as the marker for embryonic stem cells (right panel, FITC).40×

FIG. 22 is a series of photomicrographs as in FIGS. 19-21, except withSSEA-1 as the marker, which is associated with partially differentiatedcells (right panel, FITC). 40×

FIG. 23 is a series of photomicrographs taken of histological sectionsfrom hippocampus (Panel B) at day 10 following intraventricularinjection of PC of the invention in rats subjected to unilateral kainicacid lesions of the hippocampus (an animal model of epilepsy).Immunostaining with antibodies to human nestin shows that thesetransplanted PC exhibit purposeful migration to the damaged hippocampus,shown in panels A, D, E and F, and no migration to the healthy,contralateral, hippocampus (Panel C).

FIG. 24A-D is a series of photomicrographs taken of histologicalsections from hippocampus showing multi-lineage differentiation of PCimplanted in the rat model of epilepsy. Panel A shows immunostainingwith antibodies to human mitochondria at 10 days after PC injection intothe ventricle, where human PC repopulated the damaged CA3 zone of thehippocampus. Panel B shows immunostaining with antibodies to theinhibitory neurotransmitter, GABA, at 16 days after PC injection,showing that PC differentiate into inhibitory GABAergic neurons tocounteract the epileptogenic hippocampus. Panels C & D showimmunostaining with antibodies to human mitochondria at 10 days after PCinjection, showing human PC have differentiated into both neurons andastrocytes. The inserts at each panel show the contralateralhippocampus, which is free of transplanted PC.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of an unexpected type ofpluripotent cells (PC) and a culture medium optimized for long-termgrowth of these cells, which express markers of both human neuralprogenitor cells (NPC) and stem cells. The invention also relates tosuccessful cryopreservation of NPC/PC. PC cultured in accordance withthe invention are capable of surviving in vitro for longer than oneyear, and as long as three and a half years. Cryopreservation of PC inaccordance with the invention results in over 95% viability uponthawing. In addition, the invention provides variations on the culturemedium that allow for manipulation of the cultured PC to achieveattachment and differentiation when desired. PC cultured in accordancewith the invention have been successfully transplanted into the brain,providing restoration of structure and function in an animal model ofParkinson's disease and in an animal model of epilepsy. In addition, PCimplanted simultaneously with or shortly after the seizure-inducinglesion protect the brain from structural damage and prevent seizureactivity.

DEFINITIONS

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein, “low calcium” medium refers to less than 0.15 mM calcium(final concentration), and typically about 0.03-0.09 mM. Low calciummedium does not include calcium-free medium. “High calcium” mediumrefers to greater than 0.15 mM calcium.

As used herein, “neural progenitor cell” (NPC) refers to cells that areimmunopositive for nestin, capable of continuous growth in suspensioncultures and, upon exposure to appropriate conditions, can differentiateinto neurons or glial cells. A neural progenitor cell, as referred toherein, is capable of surviving for at least 2-3 years in vitro. SSEA-1is also a marker for NPC.

As used herein, “pluripotent cell” (PC; or pluripotent stem cell, PSC)refers to cells that are immunopositive for the pluripotent cellmarkers, TRA-1-60, TRA-1-81, SSEA4, and Oct-4.

As used herein, “genetically modified” refers to cells that have beenmanipulated to contain a non-native transgene by recombinant methods.For example, cells can be genetically modified by introducing a nucleicacid molecule that encodes a selected polypeptide.

As used herein, “transgene” means DNA that is inserted into a cell andthat encodes an amino acid sequence corresponding to a functionalprotein. Typically, the encoded protein is capable of exerting atherapeutic or regulatory effect.

As used herein, “protein” or “polypeptide” includes proteins, functionalfragments of proteins, and peptides, whether isolated from naturalsources, produced by recombinant techniques or chemically synthesized.Polypeptides typically comprise at least about 6 amino acids, and aresufficiently long to exert a biological or therapeutic effect.

As used herein, “vector” means a construct, which is capable ofdelivering, and preferably expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

The term “nucleic acid” or “polynucleotide” refers to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogs of natural nucleotides that hybridize to nucleic acids in amanner similar to naturally-occurring nucleotides.

As used herein, “pharmaceutically acceptable carrier” includes anymaterial which, when combined with an active ingredient, allows theingredient to retain biological activity and is non-reactive with thesubject's immune system. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Preferred diluents for aerosol orparenteral administration are phosphate buffered saline or normal (0.9%)saline.

Compositions comprising such carriers are formulated by well knownconventional methods (see, for example, Remington's PharmaceuticalSciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton,Pa., 1990).

As used herein, “a” or “an” means at least one, unless clearly indicatedotherwise.

Pluripotent Cells

The invention provides pluripotent cells (PC) that can be maintainedindefinitely in culture, stain positively for bromodeoxyuridine (BrdU),TRA-1-60, TRA-1-81, SSEA-4, SSEA-1, Oct-4 and nestin, and aremultipotent. The PC of the invention are capable of generating neurons(e.g., MAP2, neuron specific enolase or neurofilament positive cells)and glia (e.g., GFAP or galactocerebroside positive cells), as well asother cell types. PC of the invention can be maintained in cell culture,typically as a suspension culture, for at least one year. The PCdescribed herein have been maintained for as long as three years.

The PC of the invention exhibit 50% growth in the first 2 days inculture, and doubling times of less than 10 days, typically about 6days. Doubling times of as little as 5 days have been observed. Inaddition, these cells continue to grow in culture for extended periodsof time. Unlike NPC cultured in conventional media such as Neurobasal™medium, however, these cultures do not show a decline after 3-4 months,but continue to survive and expand for years, and through hundreds ofpassages.

In addition, the PC of the invention exhibit normal structure andfunction that is typical of progenitor cells. As shown in FIG. 5, PCform embryoid bodies in culture. FIG. 4 shows a confluent growth of PCthat remain undifferentiated, and FIG. 6 shows PC growing in floatingclusters. FIGS. 12 and 13 are electron micrographs, showing the normalultrastructure of PC of the invention.

PC can be prepared from mesencephalon and/or telencephalon of fetalbrain, as described in Example 1 below. Typically, the tissue isdissected in a general purpose serum-free medium, such as Hank'sBalanced Salt Solution (HBSS) with 0.25 μg/ml of Fungizone and 10 μg/mlof Gentamicin, under sterile conditions.

The cultures described herein will initially include a small percentageof Oct-4-positive cells, and mostly nestin-positive NPC cells. Over aperiod of months in culture, the proportion of Oct-4-positive cellsincreases significantly. For example, a typical culture will shift frombeing 5% Oct-4-positive cells to up to 30% Oct-4-positive cells in fourmonths.

The PC of the invention can be used in all the ways described herein forNPC. The Oct-4-positive status of these cells indicates that they arecapable of many additional uses beyond the neural environment. Thepluripotent nature of these cells make them attractive for placement ina variety of tissue environments, wherein local cytokines (naturaland/or exogenously supplied) and other signals induce appropriatedifferentiation and migration. In the description of methods thatfollows, it is understood that NPC refers to NPC and/or PC.

Media and Methods for Cell Culture

The structure and function of PC in culture is subject to manipulationvia the culture medium. For example, raising the calcium concentrationof the medium from 0.05 mM to 0.1 mM leads to attachment of theprogenitor cells to the culture flask (see FIG. 7). The addition of LIFto the culture medium extends the doubling time, but allows for a higherpopulation of neurons. Addition of LIF also helps to prevent formationof large clusters of PC. TGFα in the medium serves to significantlyreduce doubling time (e.g., from 14 days to 5 days). Accordingly, theculture medium is selected in accordance with the particular objectives,with some ingredients favoring growth and expansion and otheringredients favoring attachment and differentiation.

For general purposes, the cell culture requires a low calcium basalmedium (e.g., Ca++ free EMEM supplemented with calcium chloride),typically a B27 or equivalent supplement, and growth factors (e.g., EGF,FGF, TGFα). Optional ingredients include L-glutamine and LIF, whichpromote growth of neurons.

Example 3 below provides a detailed description of the optimization ofculture media for expansion and for differentiation of PC. In general,long-term growth and expansion requires a low calcium concentration.This is typically achieved by use of a calcium-free minimum essentialmedium (EMEM) to which calcium is added. Optimal growth and expansionhas been observed at calcium concentrations of 0.05-0.06 mM. As thecalcium concentration rises, e.g., above 0.15 mM, network formationsbetween the neurons in culture are observed as they take on a moredifferentiated neuronal phenotype. In these higher calcium cultures,only 1-2% of the cells are immunopositive for the astrocytic markerGFAP, even without the addition of LIF to the culture medium.

The following table summarizes the range of concentrations suitable forculture medium components:

Component: Concentration: B27   0.5-2.5% Calcium Chloride 0.05 mM-0.12mM Epidermal Growth Factor  15 ng/μL-100 ng/μl Basic Fibroblast GrowthFactor  10 ng/μL-150 ng/μL Transforming Growth Factor Alpha 10 ng/μL-75ng/μL Leukemia Inhibitory Factor  25 ng/μl-150 ng/μl L-Glutamine 0.1mM-0.7 mM N2 Supplement 0.3%-2.0%NPC are typically grown in suspension cultures. Initial plating ofprimary cells was optimal at 30,000 to 50,000 cells/cm². Medium changescan be made every 6 days by removing the cells to a test tube andspinning (e.g., 5 min at 1,500 rpm). Typically, all but 2 ml of theoriginal medium is discarded and the pellet is resuspended in theremaining 2 ml of original medium combined with an additional 3 ml offresh medium. When density exceeds 400,000 cells/ml, the cells can besplit into two culture vessels (e.g., T75 flasks). Trituration of thecells at the time of feeding helps to break up clusters of NPC andmaintain their suspension in the culture medium. Those skilled in theart will appreciate that variation of these parameters will be toleratedand can be optimized to suit particular objectives and conditions.

The PC of the invention can be used in therapeutic and diagnosticapplications, as well as for drug screening and genetic manipulation.The PC and/or culture media of the invention can be provided in kitform, optionally including containers and/or syringes and othermaterials, rendering them ready for use in any of these applications.

Cryopreservation of PC

The invention provides optimized methods and media for freezing andthawing of NPC and PC. The ability to store and successfully thaw NPCand PC is valuable to their utility in clinical applications andensuring a continued and consistent supply of suitable cells. While mostexperts working with progenitor and pluripotent cell populations observeonly a 2-30% survival of cells after freeze-thaw, the present inventionoffers media and methods that result in over 50% survival followingfreeze-thaw, with viability typically greater than 95%.

For cryopreservation, PC (or NPC) are suspended in a low calcium mediumsupplemented with B27 and DMSO, and the trophic factors used in theexpansion culture medium. Typically, the growth factors in thecryopreservation medium comprise about 20-100 ng/ml epidermal growthfactor (EGF); about 10-50 ng/ml fibroblast growth factor basic (bFGF);and about 1-150 ng/ml transforming growth factor-alpha (TGFα). Forthawing, both the culture medium and the flask, or other vessel intowhich the cells will be grown, are pre-warmed to 15-40° C., preferablyto approximately 25-37° C. Typically, culture flasks (or other vessel)are pre-warmed in an incubator with the same or similar gas, humidityand temperature conditions as will be used for growing the cells. Forexample, typical temperature is about 37° C., and typical CO₂ level isabout 5% (and O₂ the remaining 95%).

Therapeutic Use of Pluripotent Cells

The PC of the invention can be implanted into the central nervous system(CNS) of a host using conventional techniques. Neural transplantation or“grafting” involves transplantation of cells into the parenchyma, intothe ventricular cavities or subdurally onto the surface of a host brain.Conditions for successful transplantation include: 1) viability of theimplanted cells; 2) formation of appropriate connections and/orappropriate phenotypic expression; and 3) minimum amount of pathologicalreaction at the site of transplantation. Typically, the transplantationis by injection into the cerebral ventricles.

Therapeutic use of PC can be applied to degenerative, demyelinating,excitotoxic, neuropathic and traumatic conditions of the CNS. Examplesof conditions that can be treated via PC grafts include, but are notlimited to, Parkinson's disease (PD), Huntington's disease (HD),Alzheimer's disease (AD), multiple sclerosis (MS), amyotrophic lateralsclerosis (ALS), epilepsy, stroke, ischemia and other CNS trauma.

Methods for transplanting various neural tissues into host brains havebeen described in Neural Transplantation: A Practical Approach, S. B.Dunnett & A. Bjorklund (Eds.) Irl Pr; 1992, incorporated by referenceherein. These procedures include intraparenchymal transplantation, i.e.within the host brain (as compared to outside the brain orextraparenchymal transplantation), achieved by injection or depositionof tissue within the host brain so as to be opposed to the brainparenchyma at the time of transplantation.

The procedure for intraparenchymal transplantation involves injectingthe donor cells within the host brain parenchyma stereotactically. Thisis of importance if it is required that the graft become an integralpart of the host brain and to survive for the life of the host. In someembodiments, intraparenchymal transplantation involvespre-differentiation of the cells. Differentiation of the cells, however,limits their ability to migrate and form connections. Intraparenchymaltransplantation of pre-differentiated cells is typically preferred whenthe objective is to achieve neurochemical production at the site ofimplantation. It has been observed, however, that undifferentiated PCcan, upon implantation into the brain, differentiate as appropriate tothe environment.

Alternatively, the graft may be placed in a cerebral ventricle orsubdurally, i.e. on the surface of the host brain where it is separatedfrom the host brain parenchyma by the intervening pia mater or arachnoidand pia mater. For subdural grafting, the cells may be injected aroundthe surface of the brain. In some embodiments, the PC are injectedintravenously. PC introduced intraventricularly or intravenously willmigrate to the appropriate region on the host brain. Intraventricular(or intravenous) transplantation is preferred when the objective isrestoration of circuitry and function.

Injections into selected regions of the host brain may be made bydrilling a hole and piercing the dura to permit the needle of amicrosyringe to be inserted. The microsyringe is preferably mounted in astereotaxic frame and three dimensional stereotaxic coordinates areselected for placing the needle into the desired location of the brainor spinal cord. For grafting, the cell suspension is drawn up into thesyringe and administered to anesthetized graft recipients. Multipleinjections may be made using this procedure. Examples of CNS sites intowhich the PC may be introduced include the putamen, nucleus basalis,hippocampus cortex, striatum or caudate regions of the brain, as well asthe spinal cord and ventricles.

The cellular suspension procedure permits grafting of PC to anypredetermined site in the brain or spinal cord, is relativelynon-traumatic, allows multiple grafting simultaneously in severaldifferent sites or the same site using the same cell suspension, andpermits mixtures of cells having different characteristics. Multiplegrafts may consist of a mixture of cell types. Alternatively, the graftconsists of a substantially pure population of PC. Typically, fromapproximately 10⁴ to approximately 10⁸ cells are introduced per graft.The amount of cells used is typically constrained by volume, both interms of a suitable volume for injection and constraints of the siteinto which the cells are to be injected. An implantation of 500,000cells has been found to achieve suitable results, even where far fewercells were needed. Any excess cells are cleared from the site, and noevidence has been found of implanted cells that failed to either migrateto a site of disease or damage or be cleared. Optionally, the PC can begrafted as clusters of undifferentiated cells. Alternatively, the PC canbe induced to differentiate prior to implantation.

For transplantation into cavities, which may be preferred for spinalcord grafting, tissue is removed from regions close to the externalsurface of the CNS to form a transplantation cavity, for example byremoving glial scar overlying the spinal cord and stopping bleeding witha material such a gelfoam. Suction may be used to create the cavity. Thecell suspension is then placed in the cavity.

Grafting of PC into a traumatized brain will require differentprocedures. For example, the site of injury should be cleaned andbleeding stopped before attempting to graft. In addition, the donorcells should possess sufficient growth potential to fill any lesion orcavity in the host brain to prevent isolation of the graft in thepathological environment of the traumatized brain.

Genetically Modified PC

Although one advantage of the PC of the invention is the ability to usethem without pre-differentiation or genetic modification, these cellsare amenable to genetic modification. In some embodiments, the presentinvention provides methods for genetically modifying PC for graftinginto a target tissue site or for use in screening assays and thecreation of animal models for the study of disease conditions.

In one embodiment, the cells are grafted into the CNS to treatdefective, diseased and/or injured cells of the CNS. The methods of theinvention also contemplate the use of grafting of transgenic PC incombination with other therapeutic procedures to treat disease or traumain the CNS or other target tissue. Thus, genetically modified PC of theinvention may be co-grafted with other cells, both genetically modifiedand non-genetically modified cells, which exert beneficial effects oncells in the CNS. The genetically modified cells may thus serve tosupport the survival and function of the co-grafted, non-geneticallymodified cells.

Moreover, the genetically modified cells of the invention may beco-administered with therapeutic agents useful in treating defects,trauma or diseases of the CNS (or other target tissue), such as growthfactors, e.g. nerve growth factor (NGF), gangliosides, antibiotics,neurotransmitters, neuropeptides, toxins, neurite promoting molecules,and anti-metabolites and precursors of these molecules, such as theprecursor of dopamine, L-dopa.

Vectors carrying functional gene inserts (transgenes) can be used tomodify PC to produce molecules that are capable of directly orindirectly affecting cells in the CNS to repair damage sustained by thecells from defects, disease or trauma. In one embodiment, for treatingdefects, disease or damage of cells in the CNS, PC are modified byintroduction of a retroviral vector containing a transgene ortransgenes, for example a gene encoding nerve growth factor (NGF)protein. The genetically modified PC are grafted into the centralnervous system, for example the brain, to treat defects, disease such asAlzheimer's or Parkinson's, or injury from physical trauma, byrestoration or recovery of function in the injured neurons as a resultof production of the expressed transgene product(s) from the geneticallymodified PC. The PC may also be used to introduce a transgene product orproducts into the CNS that enhance the production of endogenousmolecules that have ameliorative effects in vivo.

Those skilled in the art will appreciate a variety of vectors, bothviral and non-viral, that can be used to introduce the transgene intothe PC. Transgene delivery can be accomplished via well-knowntechniques, including direct DNA transfection, such as byelectroporation, lipofection, calcium phosphate transfection, andDEAE-dextran. Viral delivery systems include, for example, retroviralvectors, lentiviral vectors, adenovirus and adeno-associated virus.

The nucleic acid of the transgene can be prepared by recombinant methodsor synthesized using conventional techniques. The transgene may includeone or more full-length genes or portions of genes. The polypeptidesencoded by transgenes for use in the invention include, but are notlimited to, growth factors, growth factor receptors, neurotransmitters,neuropeptides, enzymes, gangliosides, antibiotics, toxins, neuritepromoting molecules, anti-metabolites and precursors of these molecules.In particular, transgenes for insertion into NPC include, but are notlimited to, tyrosine hydroxylase, tryptophan hydroxylase, ChAT,serotonin, GABA-decarboxylase, Dopa decarboxylase (AADC), enkephalin,amphiregulin, EGF, TGF (α or β), NGF, PDGF, IGF, ciliary neuronaltrophic factor (CNTF), brain derived neurotrophic factor (BDNF),neurotrophin (NT)-3, NT-4, and basic fibroblast growth factor (bFGF).

Although those skilled in the art appreciate the advantages of usinggenetically modified PC, it is also appreciated that, in someembodiments, it is preferable to use a preparation of PC that is free ofgenetically modified cells. As described in the Examples below,transplanted PC of the invention, free of genetically modified cells orother cell types, are able to migrate to a site of damage or dysfunctionand adopt a phenotype tailored to the needs of the damaged region. Thishas been observed in both an animal model of Parkinson's disease and ananimal model of epilepsy. Accordingly, the desired therapeutic effectcan be achieved without any concerns that might be associated with useof transgenes and genetically modified cells.

Treatment includes prophylaxis and therapy. Prophylaxis or therapy canbe accomplished by a single direct injection at a single time point ormultiple time points to a single or multiple sites. Administration canalso be nearly simultaneous to multiple sites. Patients or subjectsinclude mammals. In one embodiment, the mammals are equine, canine,feline, porcine, ovine or rodent. In another embodiment, the subjectsare rodent and human. The subject is typically a human.

Administration and Dosage

The compositions are administered in any suitable manner, often withpharmaceutically acceptable carriers. Suitable methods of administeringcells in the context of the present invention to a subject areavailable, and, although more than one route can be used to administer aparticular cell composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

The dose administered to a patient, in the context of the presentinvention, should be sufficient to effect a beneficial therapeuticresponse in the patient over time, or to inhibit disease progression.Thus, the composition is administered to a subject in an amountsufficient to alleviate, reduce, cure or at least partially arrestsymptoms and/or complications from the disease or condition. An amountadequate to accomplish this is defined as a “therapeutically effectivedose.”

Routes and frequency of administration of the therapeutic compositionsdisclosed herein, as well as dosage, will vary from individual toindividual, and may be readily established using standard techniques.Typically, the pharmaceutical compositions are administered byinjection. A single injection may suffice or, in some embodiments,between 1 and 10 doses may be administered over a 52 week period.Alternate protocols may be appropriate for individual patients.

A suitable dose is an amount of a substance that, when administered asdescribed above, is capable of promoting a therapeutic response, and isat least a 10-50% improvement relative to the untreated level. Ingeneral, an appropriate dosage and treatment regimen provides thematerial in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., more frequent remissions, completeor partial, or longer disease-free survival) in treated patients ascompared to non-treated patients.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions comprising PC and,optionally, a physiologically acceptable carrier. Pharmaceuticalcompositions within the scope of the present invention may also containother compounds that may be biologically active or inactive. Forexample, one or more biological response modifiers may be present,either incorporated into a fusion polypeptide or as a separate compound,within the composition.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administration.Compositions of the present invention may be formulated for anyappropriate manner of administration, including for example,intracranial, intraventricular or subdural administration. Suchcompositions may also comprise buffers (e.g., neutral buffered saline orphosphate buffered saline), carbohydrates (e.g., glucose, mannose,sucrose or dextrans), mannitol, proteins, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.

EXAMPLES

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Example 1 Preparation of Progenitor Cells

This example demonstrates the preparation of brain progenitor cells(BPC), also referred to as neural progenitor cells (NPC). These samepreparations, initially characterized as NPC (or BPC), were laterdetermined to have features and express markers associated withpluripotent cells. The BPC were derived from the telencephalon (T lines)and mesencephalon (M lines) of fetal brain. Fetal tissue was obtainedfrom physicians in the local area using the guidelines recommended bythe National Institutes of Health. The donor was approached with therequest for tissue donation only after an elective abortion wasperformed, and informed consent was subsequently obtained. No monetarycompensation or other incentive were offered to the patient,gynecologist, or clinic. A sample of maternal blood was obtained and thefollowing serologic tests were performed: HIV, hepatitis A, B, and C,HTLV-1, VDRL, and CMV. Fetal brain tissue was obtained through alow-pressure aspiration technique under sterile conditions. There was nochange in the indication, timing, or methodology of the abortion betweenprocedures. Fetal tissue immediately adjacent to the mesencephalon wascultured for aerobic and anaerobic bacteria, HSV, and CMV. Microscopicdiagnosis was also performed using Gram stain. Fetal tissue from donorswith a history of genital herpes, cancer, asthma, lupus, rheumatoidarthritis, allergies, vasculitis of autoimmune origin, drug abuse, orprostitution was excluded.

Gestation of the fetal cadaver was determined according to crown-to-rumplength (CRL) as measured by ultrasound. The gestational age ranged from6 to 8 weeks (CRL 20 to 24 mm). The samples of telencephalon andmesencephalon were obtained from 2 donors (CRL: 20 and 24 mm).Dissections were carried out at 4° C. in a laminar flow hood(Environmental Air Control, Inc.), under a dissecting microscope (Leica,Wild MJZ, Meerbrugg, Switzerland). A general purpose serum-free medium(Ultraculture, Whittaker Bioproducts) was used, with the addition of, 5mMol of L-glutamine and 10 μg/ml of Kanamycin and 0.25 μg/ml ofFungizone. The fetal tissue was rinsed ten times with the culturemedium, and then the brain was stripped of cartilaginous skull and themeninges and transferred to Hank's Balanced Salt Solution (HBSS)supplemented with 10 μg/ml of Kanamycin sulfate and 0.25 μg/ml ofFungizone for microdissection. The dorsal cortex from both hemispheres(telencephalon) was removed parasagittally. Further, the rostral half ofventral mesencephalon and tectum was dissected. Collected samples werethoroughly minced with microscissors and triturated using sterilefire-polished pipettes. No prior trypsinization was used. Before platingcells to culture flasks or onto glass chambered slides, the cellviability (Trypan Blue exclusion test) and density were assessed.Average viability was 96%. The optimal plating density was found to be30,000 to 50,000 cells/cm².

Example 2 Characterization of Source Tissue

This example describes the characterization of tissue dissected for theabove preparation of BPC. Areas of the fetal brain tissue adjacent tothe dissected tissue were treated similarly and fixed forimmunocytochemistry and electron- and light microscopy. These adjacentsections were analyzed retrospectively for viability and functionalspecificity.

For morphological analysis, cortex and mesencephalon were taken from thefetus and processed for immunocytochemistry or ultrastructuralmorphology. Following dissection, part of the tissue was fixed in 4%buffered (pH 7.4) PFA fixative, then embedded in paraffin and sectionedon a rotary microtome. Samples of this tissue were processed in ahistochemical procedure to visualize the various neuronal and glialmarkers (AchE, TH, NSE, MAP2, BrDU, Nestin, etc.).

Immunocytochemical labeling with peroxidase reaction was carried outwith antibodies to the glial marker glial fibrillary acidic protein(GFAP; Lipshaw, Philadelphia, Pa.), the neurotransmitter GABA (SigmaChemical Co., St. Louis, Mo.), and a dopaminergic marker, thecatecholaminergic synthesis enzyme TH (Sigma Chemical Co., St. Louis,Mo.). Briefly, sections were deparaffinized and rehydrated in adescending series of ethanol baths, then incubated in 3% hydrogenperoxide blocking solution (Signet Laboratories, Dedham, Mass.). Theprimary antibody was applied onto the slides, and then removed with tworinses of phosphate-buffered saline. Slides were then incubated inlinking reagent and then labeling reagent, then visualized with AECchromogen (Signet Laboratories, Dedham, Mass.). For electron microscopy,the tissue was fixed in Karnovsky's fixative, postfixed in 1% osmiumtetroxide, dehydrated through a series of ethanols and propylene oxide,then embedded in Medcast resin (Ted Pella, Redding, Calif.). Ultrathinsections were collected on copper grids, stained with lead and uraniumand viewed with a JEOL-100CX electron microscope.

After two to four passages, most of the cultured cells were harvestedand frozen in liquid Nitrogen. Cryo medium contains the expansionculture medium with 10% DMSO, 4% of B-27 supplement, and 5 to 7 μl/ml ofMEM non-essential amino acids solution (Gibco, N.Y.).

Cells were prepared for immunocytochemical staining using conventionaltechniques as described in U.S. patent publication number 20050118561,filed Dec. 2, 2004, and published Jun. 2, 2005. Cells were stained forglial fibrillary acidic protein (GFAP), neuron specific enolase (NSE),5-Bromodeoxyuridine (BrDU), CD 34, and Leukocyte Common Antigen (CD 45).This staining protocol was also used with antibodies to Oct-4(Chemicon), beta tubulin class III (Serotec), nestin (R&D Systems),tyrosine hydroxylase (Chemicon), and human mitochondria (Chemicon).

Example 3 Optimization of Culture Media

This example describes the various media components tested for theirinfluence on expansion and differentiation of BPC. Growth rates of thetelencephalon- and mesencephalon-derived BPC were compared in threestandard culture media: Dulbecco's Modification of Eagle's Medium(DMEM); Eagle's Minimum Essential Medium (EMEM) without calcium(Biowhittaker), Neurobasal (GibcoBRL), Ultraculture (Biowhittaker), andPFMR-4+8F (BRF) with at least 25 variable combinations of mitogens bFGF,EGF, TGFα, LIF; Caspase 3 and 8 inhibitors; and B-27 supplement. Theefficacy of each combination was tested by cell viability and doublingtime during short- and long-term expansion, as well as behavioraleffects in the rat PD model after intra-striatal transplantation. TheEMEM-based, low calcium culture medium with addition of bFGF, EGF, TGFα,LIF, and B-27 presented with the best results.

After the numerous ingredients were tested, perhaps the most surprisingresult was the lack of benefit upon addition of the caspace-1 inhibitor,either acetyl-Tyr-Val-Ala-Asp (Ac-YVAD) or acetyl-Tyr-Val-Ala-Aspchloromethyl ketone (Ac-YVAD-CMK) (Calbiochem). In fact, the presence ofcaspace inhibitor in the growth medium was associated with decreasedcell counts. In addition, no benefit was observed with the use ofinterleukin-1 (IL-1). Glial cell line-derived neurotrophic factor (GDNF)and ciliary neurotrophic factor (CTNF) were both found to prompt rapiddifferentiation and cell death.

Transforming growth factor alpha (TGFα) was found to shorten doublingtime significantly (e.g., from 14 days to 5 days). Leukemia inhibitoryfactor (LIF) promoted neuronal cells and prevented the formation oflarge clusters of NPC. Basic fibroblast growth factor (bFGF) resulted ingood proliferation, even when used in the absence of other trophicfactors. Epidermal growth factor (EGF) alone did not support robustgrowth, but when combined with bFGF and TGFα, optimal growth wasobserved.

Cells grown in bFGF as the sole trophic factor were compared to NPCgrown in medium containing EGF+BFGF+TGFα (E+F+T). Two million cells peranimal were transplanted into PD rats (an animal model for Parkinson'sdisease). At 6 days post-transplant, the bFGF only cells showed a 12%decrease in density, while the E+F+T cells exhibited an increase indensity of 167%.

Progenitor Expansion Medium Basal Medium:

Eagle's Minimum Essential Medium (EMEM) without calcium, BioWhittaker,Inc., Walkersville, Md., cat #06-1746.

Supplements:

B27 (2%), Gibco BRL, cat# 17504r-hEGF (20 ng/ml), Peprotech, cat# 100-15r-hFGF basic (bFGF, FGF2), (20 ng/ml), Peprotech, cat#100-18BSodium Pyruvate (0.11 mg/ml), Sigma, cat# S-8636Calcium Chloride 2H₂O, (0.1 mM), Sigma, cat#C-7902

Optional:

Gentamicin (50 μg/ml), Sigma, cat#G-1272Amphotericin B (1.25 μg/ml), Sigma, cat#A-2942or Sigma's 100× antibiotic/antimycotic, cat#A-9909

Progenitor Differentiation Medium Basal Medium:

PFMR-4+8F, Biological Research Faculty and Facility, Inc (BRFF),cat#SF-240or DMEM, Neurobasal, or EMEM without calcium (brought up to 0.1 mMCaCl₂)

Differentiation Factors:

Glial Cell-Derived Neurotrophic Factor (GDNF) (10 ng/ml), Sigma, cat#G-1777IL-1 alpha, (100 μg/ml), Sigma, cat# 1-2778IL-11 (1 ng/ml), Sigma, cat# 1-3644Leukemia Inhibitory Factor (LIF), (1 ng/ml), Sigma, cat# L-5283N⁶,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate (db-cAMP), (100μM), Sigma, cat#D-0627Forskolin (5 μM), Calbiochem-Behring Corp, cat#344270

Optional:

0.25 μg/ml fungizone10 μg/ml kanamycin sulfate

Media Preparation:

Glutamate, when added to medium, is used only to provide for initialplating—subsequent feedings use medium without glutamate.

Expansion Medium Formulation Recipe Notes 95.5 ml basal medium 97.5 mlbasal Calcium-free EMEM preferred for medium progenitor cell expansion;for differentiation, can use EMEM, DMEM or Neurobasal 0.05 mM CaCl₂ 120μl/100 ml Only added to calcium-free EMEM; adjust quantity for expansionvs. differentiation 2% B27 supplement 2.0 ml B27 0.5 mM L-glutamine 0.25ml 200 mM Promotes growth of neurons (29.2 L-glutamine mg/ml) over glia,who prefer 2 mM L- glutamine 0.5 mM L-glutamine = 73 mg/L 100 ml med. =7.3 mg = 0.25 ml 200 mM L-glutamine 2 μg EGF (20 ng/ml) 2 × 25 μlaliquot (40 ng/μl EGF) 1 μg FGF (10 ng/ml) 1 × 25 μl aliquot (40 ng/μlFGF) 1 μg TGFα (10 ng/ml) 1 × 25 μl aliquot (40 ng/μl TGFα)

Differentiation Medium Recipe Formulation Notes 97.5 ml basal medium97.5 ml EMEM BioWhitaker w/out calcium Cat#06-174G 2.0 ml B27 2% B27supplement 1 ml 11 mg/ml Na 0.11 mg/ml sodium pyruvate 40 μl 25 mM CaCl₂0.1 mM CaCl₂ 50 μl EGF(2 aliquots @40 ng/μl) 2 μg EGF; 20 ng/ml EGF 50μl bFGF(2 aliquots@40 ng/μl) 2 μg FGF; 20 ng/ml FGF 25 μl TGFα(1 aliquotTGF@40 ng/μl) 1 μg TGF; 10 ng/ml 100 μl LIF 1 μg LIF; 10 ng/ml LIF

Neurobasal medium: Formulation Recipe Notes 97.5 ml Neurobasal medium97.5 ml Neurobasal medium 2% B27 supplement 2.0 ml B27 0.5 mML-glutamine 0.25 ml 200 mM L-glutamine Promotes growth (29.2 mg/ml) ofneurons over glia, who prefer 2 mM L-glutamine 25 μM L-Glutamic acid 184μl 2 mg/ml L-glutamic acid Helps cells attach (20 mg L-Glu + 10 mlddH₂0) 2 μg EGF (20 ng/ml) 2 × 25 μl aliquot @40 ng/μl 1 μg FGF (10ng/ml) 1 × 25 μl aliquot @40 ng/μl 1 μg TGFα (10 ng/ml) 1 × 25 μlaliquot @40 ng/μl Once made, this medium keeps 1-2 weeks refrigerated.

Example 4 Features of Cells Cultured in Media of the Invention

The PC cultured in the medium of the invention have been shown to havethe characteristics of neural progenitor cells: they can be maintainedindefinitely in EMEM culture, show positive staining for BrDU, expressNestin, under low [Ca++] conditions they are capable of generatingdopaminergic (35-60%) and serotonergic (24-40%) neurons as well as anumber of other MAP2 positive cells (10-12%), and glia (GFAP positivecells 15-23%). They also sporadically generate nucleated red cells(2-3%) in vitro and myoblasts when injected into the ischemic rat heart.

In contrast, PC will remain in suspension and undifferentiated whencultured in the low calcium medium EMEM of the invention. As the calciumconcentration is raised, e.g., to 0.1 mM, then the PC form networks andexhibit a neuronal phenotype. Even without the addition of LIF to favorneurons over glia, only 1-2% of these cultured cells are immunopositivefor the glial marker GFAP, suggesting that the population is primarilyneuronal.

Example 5 Transplantation of PC into Brain in an Animal Model ofParkinson's Disease

This example demonstrates that PC prepared in accordance with theinvention can be successfully grafted into rat brain. The example showsthat grafted cells can exhibit normal differentiation into tyrosinehydroxylase (TH) positive cells. In addition, the results show that thegrafted PC ameliorate the behavioral deficit characteristic of thisanimal model of Parkinson's disease.

For implantation, free-floating PC are removed from the culture flaskand spun as is done for medium changes. The pellet is re-suspended inthe remaining 2 mls of medium, and this concentrated suspension iscounted on a hemacytometer. Additional medium is added to bring thefinal cell concentration to 350,000 cells/μl.

The substantia nigra was lesioned via injection of 4 μl (8 μg)6-hydroxydopamine, 6-OHDA (Research Biomedicals International, Mass.)using a Hamilton syringe (Hamilton Co., Nev.). The injection was carriedout over 2 minutes, with a three minute wait after injection to allowdiffusion before removal of the needle.

Two weeks following nigral lesion, rats were placed under generalanesthesia (Ketamine 87 mg/kg and Xylazine 10 mg/kg; or 4% isofluranegas) and fixed in a stereotaxic apparatus. The scalp incision was madeand a hole was drilled in the skull at the coordinates of the striatum.The progenitor cells were implanted using a Hamilton syringe (70,000cells/2 μl per animal) into the striatum ipsilateral to the 6-OHDAlesion, at stereotaxic coordinates A=−0.11; L=3.8; V=4.5. The incisionwas then closed and treated with Betadine. All PCs were implantedwithout prior conditioning.

For rotational behavior testing, rats were injected subcutaneously withamphetamine or vehicle. Immediately after injection, animals were placedin a locomotor chamber measuring 3 feet by 3 feet (Columbus Instruments,Columbus, Ohio). Following a two-minute adjustment period, all rotationswere tracked by a CCD camera mounted over the chamber and analyzed bythe Videomex V™ video image analyzer (Columbus Instruments, Columbus,Ohio). Locomotor activity and rotation were recorded for 60 minutes.

Both groups of animals that received T5 or M5 cells showed significantand comparable reduction in their rotational behavior. In both groups ofanimals, about 14-24% of the PCs differentiated into TH-positive cells.

Example 6 Cells Implanted in Substantia Nigra Become TyrosineHydroxylase Positive

PC, both M5 and T5 cells, were implanted using a method similar to thatdescribed in Example 5 above. The M5 cell population, derived frombrainstem, was 24-30% positive for tyrosine hydroxylase (TH) prior toimplantation. After implantation, 54% of the M5 NPC were TH positive.The T5 cells, derived from forebrain, were all TH negative in culture.Once implanted, 32% of the implanted PC were TH positive.

Example 7 Differentiation of PC

Culture conditions as described above were varied and manipulated todetermine the optimal conditions to induce differentiation of PC. Theresulting optimized differentiation medium contains 0.15 mM Ca++, 0.5 mML-glutamine, 10 ng/ml GDNF, 15 ng/ml retinoic acid.

Example 8 Cryopreservation of PC

Media ingredients were varied and manipulated to determine the optimalconditions for cryopreservation of PC. B27, in addition to DMSO, appearsto provide a significant protective effect contributing to theexceptionally high viability observed in thawed PC.

For cryopreservation, PC were suspended in a low calcium medium (0.06 mMCa++EMEM) supplemented with 2% B27, LIF (15 ng/ml), EGF (50 ng/ml), FGFand TGF (25 ng/ml) and 10% DMSO. The cells are first placed in a freezerat about −40° C. for 1 to 1.5 hours, after which they are stored inliquid nitrogen. Cells can be stored at below about −80° C., typicallyat about −200° C. The liquid nitrogen storage tank used in these studiesis maintained at −197° C.

For thawing, both the culture medium and the flask was pre-warmed to 37°C. in a water bath at 37° C. Using this cryopreservation method, over95% viability is consistently observed in the PC upon thawing (using dyeexclusion cell counts). Typically, the cells appear shrunken and ofabnormal morphology for the first 5-7 days after thawing. Despite thisappearance, the cells are able to exclude trypan blue dye. After aboutone week, the cells recover to their pre-freezing state, exhibitingtypical morphology, growth and doubling times.

Example 9 Pluripotent Cells in Cultures of the Invention

Cells cultured as described above for NPC have been evaluated forexpression of the stem cell marker Oct-4. Oct-4 (“octamer-4”) is atranscription factor that is specifically expressed in embryonic andadult stem cells and tumor cells, but not in cells of differentiatedtissues (Tai et al., Carcinogenesis, published online Oct. 28, 2004).Oct-4-positive cells are also capable of developing in culture intooogonia that enter meiosis, recruit adjacent cells to form follicle-likestructures, and later develop into blastocysts (Hubner, K. et al.,Science, 2003, 300(5623):1251-6). This capacity for oogenesis in culturemakes them useful for nuclear transfer and manipulation of the germline, and as well as to create models for studies on fertility treatmentand germ and somatic cell interaction and differentiation.

Cells cultured as described above for NPC, by six weeks in culture, willshow some stem cells (Oct-4-positive), and mostly nestin-positiveprogenitor cells. Over a period of four months in culture, thepopulation shifted from containing about 5% Oct-4-positive cells toabout 30% Oct-4-positive cells. This observation could indicate thatthese cells de-differentiate in long-term culture. Alternatively, thismay reflect a selective survival of stem cells in long-term culture.

Oct-4-positive cells were also observed to co-express the NPC marker,nestin, as shown in FIG. 17. Nestin-positive cells are thus capable ofdifferentiating into neural cells, but not necessarily committed to thispath.

Example 10 Implanted PCs Restore Function in Animal Model of Parkinson'sDisease

Nigral lesions were performed in rats as described above in Example 5 tocreate the rotational behavior deficit characteristic of this rat modelof Parkinson's disease. 500,000 human NPC prepared as described abovewere injected into the cerebral ventricle. After completion ofrotational behavior studies, which confirmed successful amelioration ofrotational behavior, tissues sections were prepared forimmunohistochemical examination. Human cells from the implanted PCs werefound to have migrated to neural structures including the striatum,substantia nigra and hippocampus, and to differentiate into neurons andglia.

FIG. 18 is a photomicrograph showing an amber-brown human neuron withthe branching extensions at the center of the picture and a glial cellat the right lower corner of the picture in the rat putamen. These cellsmigrated from the cerebral ventricle of the animal that showed a 70%improvement in its rotational behavior 4 months after theintraventricular injection of 500,000 undifferentiated neural progenitorcells. Anti-human mitochondrial antibodies. 40×.

Example 11 Co-Expression of Markers for NPC and Pluripotent Cells

This example demonstrates that cells derived from human fetal forebrainand propagated in vitro as described in the preceding examples expressfeatures of both neural progenitor cells and pluripotent cells. Thecells were immunostained with antibodies directed against nestin, amarker for partially differentiated neural progenitor cells, and with asecond antibody directed against a marker for pluripotent embryonic stemcells. Four different markers were tested: TRA-1-60, TRA-1-81, SSEA-4(pluripotent cell markers) and SSEA-1 (progenitor cell marker). All fourwere found co-expressed in the same cells with nestin. Representativeimages showing double-stained cells are presented in FIGS. 19-22. Thenuclear stain DAPI was used to reference cell location, size and shape.

The cell populations of the invention have been cultured for over threeyears, and the proportion of various markers expressed in thesepopulations have been found to shift toward markers associated withundifferentiated cells over time. The percentage of cells expressingthese markers as of year 1, year 2 and year 3 in culture is shown in thefollowing table.

Cell Marker Year 1 Year 2 Year 3 Neuron Specific Enolase (NSE) 32% 15% 7% Tyrosine Hydroxylase (TH) 29%  7%  2% Glial Fibrillary AcidicProtein (GFAP) 55%  1%  1% GABA  2%  3%  2% Nestin 68% 99% 99% Oct-4 16%64% 98% Human Mitochondria 100%  100%  100%  Telomerase (hTERT) 98% p5358%

Example 12 Pluripotent Cells Transplanted to Ventricle Migrate toDamaged Hippocampus

This example demonstrates the damage-specific migration of transplantedpluripotent cells. Kainic acid was injected unilaterally into rathippocampus in accordance with conventional protocol for this animalmodel of epilepsy. The contralateral hippocampus served as anintra-subject control. Pluripotent cells of the invention (500,000 or1,000,000 cells per subject) were injected into the ventricle. After 10days, hippocampal tissue sections were obtained and stained usinganti-human nestin. As illustrated in FIG. 23, the transplanted cellswere found to migrate to the damaged hippocampus and not to thecontralateral healthy hippocampus.

Procedure 1: Excitotoxic Lesion of the Dopaminergic Neurons.

Under general anesthesia (Ketamine 87 mg/kg and Xylazine 10 mg/kg orIsofluorane gas at 4% for induction and 1.5% for maintenance), femaleSprague-Dawley rats were placed into a stereotaxic apparatus. The headsof the subjects were shaved using an electric razor, and the area to beincised was cleaned with betadine. An incision was made in the scalp, ahole was drilled in the skull, and the rat received a stereotacticinjection of 6-OHDA into the substantia nigra. Stereotaxic coordinatesfrom bregma midline and dura with incisor bar +3.3: A=5.0, L=2.0, V=8.0.The incision was then closed with staples and treated with Betadine;instruments were sterilized with alcohol between each use. Rats weregiven 1 cc of subcutaneous sterile saline, placed on a heating pad andmonitored closely until they woke up. Rats were monitored daily for oneweek post-op during their recovery, and given additional injections ofsubcutaneous saline if dehydration was observed.

Procedure 2: Kainic Acid Injection

Adult male (150-250 gm) Sprague Dawley rats were anesthetized usingKetamine 87 mg/kg and Xylazine 10 mg/kg or Isofluorane gas at 4% forinduction and 1.5% for maintenance), and unilaterally injected withkainic acid (KA) (0.4 μg/0.2 μl normal saline) in the right posteriorhippocampus [anteroposterior (AP), −5.6 mm; mediolateral (ML) 4.0 mm;dorsoventral (DV) 7.0 mm. Beginning 2-3 months after injection, ratswere observed during repeated 16-24 hr video monitoring periods for 1-2weeks to detect spontaneous behavioral seizures.

Procedure 3: Grafting Cells to the Striatum, Hippocampus, or LateralVentricles of Adult Rats

Two to three weeks following nigral and hippocampal lesion (above), ratswere again placed under general anesthesia (see procedure 1 and 2) andfixed in a stereotaxic apparatus. The scalp incision was re-opened and asecond hole drilled in the skull at the coordinates of the striatum. Thepluripotent cells were implanted into the striatum, hippocampus or brainventricles ipsilateral to the 6OHDA lesion, at stereotaxic coordinatesAnterior-Posterior (AP) −0.11; Medial-Lateral (ML) 3.8 mm; Vertical (V)−4.5 mm for the striatum; AP −5.6 mm; ML 4.0 mm; V 7.0 mm for thehippocampus AP −0.8 mm; ML −1.5 mm; V −4.5 mm, and for the lateral brainventricle AP −1.0 mm, ML −1.8 mm, V −3.5 mm.

Example 13 Multi-Lineage Differentiation of Pluripotent CellsTransplanted into Hippocampus

This example demonstrates that human pluripotent cells of the inventionwill repopulate the damaged CA3 zone of the hippocampus of kainicacid-treated rats, and that these transplanted cells will differentiateinto neurons and astrocytes. FIGS. 24A, C and D show the transplantedcells at 10 days after injection. The presence of these human cells inthe CA3 zone is shown in FIG. 24A using anti-human mitochondriastaining. By 16 days after injection, transplanted cells havedifferentiated into GABAergic neurons, as shown in FIG. 24B. Thepresence of this inhibitory neurotransmitter indicates that thetransplanted cells have differentiated into a phenotype that willcounteract the excessive excitatory activity of the epileptogenichippocampus.

The presence of both neurons (FIG. 24C) and astrocytes (FIG. 24D)indicates that the transplanted cells (identified by anti-humanmitochondria staining) have differentiated into different cellpopulations by 10 days after transplantation. As shown in the insets ofFIGS. 24A-D, the contralateral, intact side of the brain is free of thetransplanted pluripotent cells.

Example 14 Implanted Cells Restore Structure of Lesioned Brain and EndSeizure Activity

This example demonstrates that human pluripotent cells of the inventionwill repopulate the brain after extensive damage induced by kainic acidlesions. In addition, lesion-induced seizure activity stopped insubjects receiving intraventricular injection of pluripotent cells.

Rats were lesioned with kainic acid as described in Example 13. Thistreatment obliterates approximately one third of the brain volume,creating a large cavity in the cerebrum, and leaves the subjectexperiencing massive and repeated seizure activity. In this study, of 48subjects receiving these lesions, 24 received intraventricular injectionof 500,000 pluripotent cells of the invention six months following thekainic acid lesion. The remaining 24 subjects received controltreatment.

Seizure activity was recorded for 12 hours daily. Essentially,implantation of pluripotent cells into the ventricles of KA-lesionedrats resulted in a shift from a chronic disease condition to an acutecondition. A further group of rats was then implanted with pluripotentcells concurrently with KA-lesion. In these subjects, no cerebral cavitywas observed, and no seizure activity developed. In other words,pluripotent cells were able to prevent the massive kainic acid-inducedcavity formation and seizures. This remarkable protective effect ofpluripotent cells indicates that administration of the cells is mostoptimal if performed shortly after a seizure has occurred, therebyminimizing the cerebral damage caused by the seizure activity and hencereducing further seizure activity resulting from seizure-induced damage.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A plurality of isolated mammalian cells that are immunopositive fornestin and for a marker of pluripotent cells, wherein the cells aresuspended in a culture medium that has a total calcium concentration of0.03 to 0.15 mM, and wherein the cells continue to proliferate in anundifferentiated state for at least 4 months.
 2. The cells of claim 1,wherein the marker of pluripotent cells is selected from the groupconsisting of TRA-1-60, TRA-1-81 and SSEA-4.
 3. The cells of claim 1,wherein the cells have a doubling rate of less than 12 days.
 4. Thecells of claim 1, wherein the cells have a doubling rate of about 5days.
 5. The cells of claim 1, wherein the cells continue to proliferatefor at least 2 years in vitro.
 6. The cells of claim 1, wherein thecells are obtained from fetal forebrain.
 7. The cells of claim 1,wherein the medium has a total calcium concentration of less than 0.1mM.
 8. The cell culture of claim 7, wherein the total calciumconcentration is about 0.05 mM.
 9. The cells of claim 1, wherein themedium further comprises: (a) about 15-100 ng/μl epidermal growth factor(EGF); (b) about 10-150 ng/μl basic fibroblast growth factor (bFGF); (c)about 10-75 ng/μl transforming growth factor-alpha (TGFα).
 10. The cellsof claim 9, further comprising: (d) about 25-150 ng/μl leukemiainhibiting factor (LIF).
 11. The cells of claim 10, wherein the LIF isabout 25 ng/μl.
 12. The cells of claim 9, wherein the EGF is about 20ng/μl.
 13. The cells of claim 9, wherein the bFGF is about 10 ng/μl. 14.The cells of claim 9, wherein the TGFα is about 10 ng/μl.
 15. The cellsof claim 9, wherein the culture medium is free of a feeder layer. 16.The cells of claim 9, wherein the culture medium is serum-free.
 17. Thecells of claim 9, further comprising 0.5-2.5% B27 supplement.
 18. Thecells of claim 9, wherein the growth factors EGF, bFGF and TGFα arerecombinant growth factors.
 19. The cells of claim 9, wherein the cellsand the growth factors are human.
 20. The cells of claim 9, wherein theculture medium further comprises about 0.11 mg/ml sodium pyruvate.
 21. Amethod of propagating pluripotent cells, comprising culturing the cellsof claim
 1. 22. The method of claim 21, wherein the culturing is in theabsence of a feeder layer.
 23. A method of transplanting humanpluripotent cells to a mammalian host, comprising: (a) obtaining thecells of claim 1; and (b) transplanting the cells to the host.
 24. Amethod of reducing seizure activity in a mammalian subject comprising:(a) identifying a mammalian subject who has had a seizure within thepreceding week; and (b) implanting the cells of claim 1 into a cerebralventricle of the host.
 25. The method of claim 24, wherein the subjecthas had a seizure within the preceding 48 hours.